Figure 8.

MPA technology principles: probes and melting curves. (a) Probes consist of a target-hybridising oligonucleotide (THO) labelled with a fluorophore (F) at the 5′end and quencher (Q) at the 3′end; the THO is shown hybridised to a partially complementary oligonucleotide (PCO). Mismatches are represented by red dotted lines. (b) In aqueous solution and low temperature the annealed THO: PCO pair is fluorescent due to the relatively high atomic distance between the fluorophore and the quencher. Exposure to increased temperature leads to thermal dissociation of the hybrid reaching a maximum rate at the corresponding dissociation temperature (T_m). As the THO becomes detached, it assumes a randomly coiled conformation causing a drop in atomic distance between fluorophore and quencher and a corresponding decrease in fluorescence. Thus the melting curve shows decreased fluorescence emission with increasing temperature. (c) The negative derivative plot of the emission reading versus temperature reveals a positive value of the melting peak at the corresponding Tm.